1. Field of the Invention
The present invention relates to new biomaterials, i.e., bonding solutions for granular bone replacements and bone replacements in the form of pastes, comprising hyaluronic acid and hyaluronic acid derivatives.
2. Description of Related Art
Hyaluronic acid
Hyaluronic acid is a natural polysaccharide composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucuronic acid. It is a linear polymer with a wide molecular weight range, depending on the source from which it is obtained, how it is prepared, and how the molecular weight is determined. In nature, it is present in the pericellular gel, in the fundamental substance of the connective tissues of vertebrates, of which it is the main component, in the synovial fluid of joints, in the vitreous humor, in umbilical cord tissues, and in the combs of domestic fowl.
Specific hyaluronic acid fractions with definite molecular weights are known which do not possess inflammatory activity, and which can therefore be used to enhance wound healing or substitute for endobulbar fluids, or which can be used in therapy for joint pathologies by intraarticular injection, as described in European Patent No. 0138572, granted to Applicants.
Also known are hyaluronic acid esters wherein all or part of the carboxy groups of the acid are esterified, and their use in the fields of pharmaceuticals, cosmetics, and biodegradable plastic materials, as described in U.S. Pat. Nos. 4,851,521 and 4,965,353 also granted to Applicants.
It is known that hyaluronic acid plays a fundamental role in tissue repair processes especially in the first stages of granulation tissue formation, stabilizing the coagulation matrix and controlling degradation, favoring the recruitment of inflammatory cells such as polymorphonucleocytes and monocytes, of mesenchymal cells such as fibroblasts and endothelial cells, and directing the subsequent migration of epithelial cells.
It is known that the application of hyaluronic acid solutions to bedsores, wounds and burns accelerates healing. The role of hyaluronic acid in the various stages of tissue repair has been described, by constructing a theoretical model, by Weigel P. H. et al.: xe2x80x9cA model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing,xe2x80x9d J. Theor. Biol., 119: 219, 1986.
Granular Bone Replacements
Granular bone replacements have been widely studied and used in dentistry and medicine owing to their biocompatible and osteoconductive properties, and because they easily fill cavities of various forms, such as those caused by alveolar bone shrinkage, postextraction cavities, and cystic cavities. The main difficulty when using this material is caused by its lack of binding properties, so that it easily becomes dislodged either during or after application. To overcome this drawback, various types of bonding materials have been proposed with which to prepare pastes containing bone granules. Fibrin is already being used, as described in patent applications JP A 60254640 and JP A 60254641.
One disadvantage of using fibrin as a bonding agent is that it can cause infection by the hepatitis virus, as well as by H.I.V. and other viruses, due to its human origin. Alternative materials have therefore been proposed, such as pullulan, chitin, glycol, carbomethylene chitin, and pectin, as described in patent application EP 0416398.
An object of the present invention is to provide a viscous solution composed of hyaluronic acid and/or hyaluronic acid esters or salts of hyaluronic acid in association with antibiotics, used singly or in combination, to bind bone replacements in granular form for use in dentistry or surgery of all kinds. Such solutions have excellent biocompatible and bioabsorbable characteristics, and are not liable to cause problems such as infection.
Another object of the present invention is to provide a paste comprising a viscous solution of hyaluronic acid and/or hyaluronic acid esters or salts of hyaluronic acid in association with antibiotics, used singly or in combination, and bone granules. The granules incorporated in the paste are firmly adhered together, thus facilitating their use in dentistry and bone surgery.
Further scope of the applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following detailed description is provided to aid those skilled in the art in practicing the present inventions Even so, the following detailed description should not be construed to unduly limit the present invention, as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.
The contents of each of the references cited herein are incorporated by reference in their entirety.
The foregoing objects are achieved by dissolving hyaluronic acid, esters of hyaluronic acid, or salts of hyaluronic acid in association with antibiotics, used singly or in combination, in water, to form highly viscous solutions. The viscosity of such solutions is at least 15 Pa.s, preferably over 22 Pa.s, at a temperature of 25xc2x0 C. and 50%xc2x15% relative humidity. The nearer the viscosity to the lower limit, the more liquid the solution; the greater the viscosity, the denser the solution. The correct degree of viscosity for ideal working conditions is a subjective parameter which depends on the individual, who can alter its viscosity by varying the concentration of the solution. The properties of the material comprising the solution, such as its molecular weight, will be selected according to the required degree of viscosity.
The solution according to the present invention is prepared by solubilizing hyaluronic acid, the partial ester of hyaluronic acid or a mixture of the same, or a salt of hyaluronic acid in association with an antibiotic or a mixture of the same, previously sterilized with gamma rays, in sterile water or buffer.
The esters of hyaluronic acid that can be used in the present invention are described in U.S. Pat. Nos. 4,851,521 and 4,965,353, and in PCT publication WO 92-13579. the salts of hyaluronic acid in association with antibiotics that can be used in the present invention are descried in U.S. Pat. No. 5,166,331. these can be used singly or in various combinations with one another, or with hyaluronic acid.
The powder is placed in the solubilization vessel under a sterile hood at a temperature of 25xc2x0 C.xc2x12xc2x0 C. and 50%xc2x15% humidity. Solubilization can be achieved in a mixer composed of two spiral elements turning in opposite directions.
Operative conditions depend on the desired viscosity and can be as follows:
All air is eliminated from the solution before use by placing it in a vacuum for two hours (minimum 0.01 mbar). It is possible to sterilize a solution prepared with non-sterile material by filtering it through a filter with a pore size of 0.22 xcexcm. In this case, to facilitate filtration, the solution can first be prepared at a lower viscosity than will be required for its application. It is then filtered and subsequently distilled under vacuum until it reaches the concentration corresponding to the desired viscosity. Solutions thus prepared can be mixed with bone granules to form a paste to be used to fill bone cavities and defects. The ratio between the quantity of solution and the quantity of granules is 1:3, w/w, or more. If the quantity of solution is too small, the granules will not be sufficiently bound together, so more solution rust be added. If the paste is too liquid for easy application, it can be placed in a high vacuum for about two minutes, repeating this operation until the correct consistency is obtained.
The bone granules that can be used in the present invention are not particularly limited. In general, it is possible to use those already in common use. Examples of these can be found in U.S. Pat. Nos. 4,693,986 and 4,629,464. The diameter of the granules can be between 50 xcexcm and 5 mm, and they can be porous or nonporous. Among the materials preferred due to their biocompatibility are granules of hydroxyapatite, tricalcium phosphate, and calcium carbonate.
For purely illustrative purposes, described hereafter are some examples of preparations of solutions and pastes according to the present invention.
The Esters of Hyaluronic Acid
Esters of hyaluronic acid useful in the present invention are esters of hyaluronic acid with aliphatic, araliphatic, cycloaliphatic or heterocyclic alcohols, in which are esterified all (so-called xe2x80x9ctotal estersxe2x80x9d) or only a part (so-called xe2x80x9cpartial estersxe2x80x9d) of the carboxylic groups of the hyaluronic acid, and salts of the partial esters with metals or with organic bases, biocompatible or acceptable from a pharmacological point of view.
The useful esters include esters which derive from alcohols which themselves possess a notable pharmacological action. The saturated alcohols of the aliphatic series or simple alcohols of the cycloaliphatic series are useful in the present invention.
In the above mentioned esters in which some of the carboxylic acid groups remain free (i.e., partial esters), these may be salified with metals or organic bass, such as with alkaline or alkaline earth metals or with ammonia or nitrogenous organic bases.
Most of the esters of hyaluronic acid (xe2x80x9cHYxe2x80x9d), unlike HY itself, present a certain degree of solubility in organic solvents. This solubility depends on the percentage of esterified carboxylic groups and on the type of alkyl group linked with the carboxyl. Therefore, an HY compound with all its carboxylic groups esterified presents, at room temperature, good solubility for example in dimethylsulfoxide (the benzyl ester of HY dissolves in DMSO in a measure of 200 mg/ml). Most of the total esters of HY present also, unlike HY and especially its salts, poor solubility in water and are essentially insoluble in water. The solubility characteristics, together with particular and notable viscoelastic properties, make the HY esters particularly preferred for use in composite membranes.
Alcohols of the aliphatic series to be used as esterifying components of the carboxylic groups of hyaluronic acid for use in composite membranes according to the present invention are for example those with a maximum of 34 carbon atoms, which may be saturated or unsaturated and which may possibly also be substituted by other free functional or functionally modified groups, such as amine, hydroxyl, aldehyde, ketone, mercaptan, or carboxyl groups or by groups derived from these, such as hydrocarbyl or di-hydrocarbylamine groups (from now on the term xe2x80x9chydrocarbylxe2x80x9d will be used to refer not only to monovalent radicals of hydrocarbons such as the CnH2n+1 type, but also bivalent or trivalent radicals, such as xe2x80x9calkylenesxe2x80x9d CnH2n or xe2x80x9calkylidenesxe2x80x9d CnH2n), ether or ester groups, acetal or ketal groups, thioether or thioester groups, and esterified carboxyl or carbamide groups and carbamide substituted by one or more hydrocarbyl groups, by nitrile groups or by halogens.
Of the above mentioned groups containing hydrocarbyl radicals, these are preferably lower aliphatic radicals, such as alkyls, with a maximum of 6 carbon atoms. Such alcohols may also be interrupted in the carbon atom chain by heteroatoms, such as oxygen, nitrogen and sulfur atoms. Preferred are alcohols substituted with one or two of the said functional groups.
Alcohols of the above mentioned group which are preferably used are those with a maximum of 12, and especially 6 carbon atoms, and in which the hydrocarbyl atoms in the above mentioned amine, ether, ester, thioether, thioester, acetal, ketal groups represent alkyl groups with a maximum of 4 carbon atoms, and also in the esterified carboxyl or substituted carbamide groups the hydrocarbyl groups are alkyls with the same number of carbon atoms, and in which in the amine or carbamide groups way be alkylenamine or alkylencarbamide groups with a maximum of 8 carbon atoms. Of these alcohols, specifically preferred are saturated and non-substituted alcohols, such as the methyl, ethyl, propyl, and isopropyl alcohols, normal butyl alcohol, isobutyl alcohol, tertiary butyl alcohol, the amyl, pentyl, hexyl, octyl, nonyl and dodecyl alcohols and, above all, those with a linear chain, such as normal octyl and dodecyl alcohols. Of the substituted alcohols of this group, the bivalent alcohols are useful, such as ethyleneglycol, propyleneglycol and butyleneglycol, the trivalent alcohols such as glycerine, the aldehyde alcohols such as tartronic alcohol, the carboxylic alcohols such as lactic acids, for example glycolic acid, malic acid, the tartaric acids, citric acid, the aminoalcohols, such as normal aminoethanol, aminopropanol, normal aminobutanol and their diaethylated and diethylated derivatives in the amine function, choline, pyrrolidinylethanol, piperidinylethanol, piperazineylethanol and the corresponding derivatives of normal propyl or normal butyl alcohol, monothicethyleneglycol or its alkyl derivatives, such as the ethyl derivative in the mercaptan function.
Of the higher saturated aliphatic alcohols, preferred are cetyl alcohol and myricyl alcohol, but for the aim of the present invention the higher unsaturated alcohols with one or two double bonds, are especially important, such as especially those contained in many essential oils and with affinity to terpene, such as citronellol, geraniol, nerol, nerolidol, linalool, farnesol, phytol. of the unsaturated lower alcohols it is necessary to consider allyl alcohol and propargyl alcohol. Of the araliphatic alcohols, preferred are those with only one benzene residue and in which the aliphatic chain has a maximum of 4 carbon atoms, which the benzene residue can be substituted by between 1 and 3 methyl or hydroxyl groups or by halogen atoms, especially by chlorine, bromine and iodine, and in which the aliphatic chain may be substituted by one or more functions chosen from the group containing fee amine groups or mono- or dimethylated or by pyrrolidine or piperidine groups. Of these alcohols, most preferred are benzyl alcohol and phenetyl alcohol.
The alcohols of the cycloaliphatic or aliphatic-cycloaliphatic series may derive from mono- or polycyclic hydrocarbons, may preferably have a maximum of 34 carbon atoms, may be unsubstituted and may contain one or more substituents, such as those mentioned above for the aliphatic alcohols. Of the alcohols derived from cyclic monoannular hydrocarbons, preferred are those with a maximum of 12 carbon atoms, the rings with preferably between 5 and 7 carbon atoms, which may be substituted for example by between one and three lower alkyl groups, such as methyl, ethyl, propyl or isopropyl groups. As specific alcohols of this group the following are most preferred: cyclohexanol, cyclohexanediol, 1,2,3-cyclohexanetroil and 1,3,5-cyclohexanetriol (phloroglucitol), inositol, and the alcohols which derive fron p-methane such as carvomenthol, menthol, and xcex1-xcex3terpineol, 1-terpineol, 4-terpineol and piperitol, or the mixture of these alcohols known as xe2x80x9cterpineolxe2x80x9d, 1,4- and 1,8 terpin. Of the alcohols which derive from hydrocarbons with condensed rings, such as those of the thujane, pinane or comphane, the following are preferred: thujanol, sabinol, pinol hydrate, D and L-borneol and D and L-isoborneol.
Aliphatic-cycloaliphatic polycyclic alcohols to be used for the esters of the present invention are sterols, cholic acids and steroids, such as sexual hormones and their synthetic analogues, especially corticosteroids and their derivatives. It is therefore possible to use: cholesterol, dihydrocholesterol, epidihydrocholesterol, coprostanol, epicoprostanol, sitosterol, stigmasterol, ergosterol, cholic acid, deoxycholic acid, lithocholic acid, estriol, estradiol, equilenin, equilin and their alkylate derivatives, as well as their ethynyl or propynyl derivatives in position 17, such as 17xcex1-ethynl-estradiol or 7xcex1-methyl-17xcex1-ethynyl-estradiol, pregnenolone, pregnanediol, testosterone and its derivatives, such as 17xcex1-methyltestosterone, 1,2-dehydrotestosterone and 17xcex1-methyl-1,2-dehydrotesterone, the alkynylate derivatives in position 17 of testosterone and 1,2-dehydrotestosterone, such as 17xcex1-ethynyltestosterone, 17xcex1-propynyltestosterone, norgestrel, hydroxyprogesterone, corticosterone, deoxycorticosterone, 19-nortestosterone, 19-nor-17xcex1-methyltestosterone and 19-nor-17xcex1-ethynyltestosterone, antihormones such as cyproterone, cortisone, hydrocortisone, prednisone, prednisolone, fluorocortisone, dexamethasone, betametbasone, paramethasone, flumethasone, fluocinolone, fluprednylidene, clobetasol, beclomethasone, aldosterone, deoxycorticosterone, alfaxolone, alfadolone, and bolasterone. As esterifying components for the esters of the present invention the following are useful: genins (aglycons) of the cardioactive glucosides, such as digitoxigenin, gitoxigenin, digoxigenin, strophanthidin, tigogenin and saponins.
Other alcohols to be used according to the invention are the vitamin ones, such as axerophthol, vitamins D2 and D3, aneurine, lactoflavine, ascorbic acid, riboflavine, thiamine, and pantothenic acid.
Of the heterocyclic acids, the following can be considered as derivatives of the above mentioned cycloaliphatic or aliphatic-cycloaliphatic alcohols if their linear or cyclic chains are interrupted by one or more, for example by between one and three heteroatoms, for instance chosen from the group formed by xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N, and xe2x80x94NHxe2x80x94, and in these, there may be one or more unsaturated bonds, for example double bonds, in particular between one and three, thus including also heterocyclic compounds with aromatic structures. For example the following should be mentioned: furfuryl alcohol, alkaloids and derivatives such as atropine, scopolamine, cinchonine, 1a cinchonidine, quinine, morphine, codeine, nalorphine, N-butylscopolammonium bromide, ajmaline; phenylethylamines such as ephedrine, isoproterenol, epinephrine; phenothiazine drugs such as perphenazine, pipothiazine, carphenazine, homofenazine, acetophenazine, fluophenazine, and N-hydroxyethylpromethazine chloride; thioxanthene drugs such as flupenthixol and clopenthixol; anticonvulsants such as meprophendiol; antipsychotics such as opipramol; antiemetics such as oxypendyl; analgesics such as carbetidine and phenoperidine and methadol; hypnotics such as etodroxizine; anorexics such as benzidrol and diphemethoxidine; minor tranquilizers such as hydroxyzine; muscle relaxants such as cinnamedrine, diphylline, mephenesin, methocarbamol, chlorphenesin, 2,2-diethyl-1,3-propanediol, guaifenesin, hydrocilamide; coronary vasodilators such as dipyridamole and oxyfedrine; adrenergic blockers such as propanolol, timolol, pindolol, bupranolol, atenolol, metroprolol, practolol; antineoplastics such as 6-azauridine, cytarabine, floxuridine; antibiotics such as chloramphenicol, thiamphenicol, erythromycin, oleandomycin, lincomycin; antivirals such as idoxuridine; peripheral vasodilators such as isonicotinyl alcohol; carbonic anhydrase inhibitors such as sulocarbilate; antiasthmatic and antiinflammatories such as tiaramide; sulfamidics such as 2-p-sulfanilonoethanol.
In some cases hyaluronic acid esters may be of interest where the ester groups derive from two or more therapeutically active hydroxylic substances, and naturally all possible variants may be obtained. Especially interesting are the substances in which two types of different ester groups deriving from drugs of a hydroxylic character are present and in which the remaining carboxyl groups are free, salified with metals or with a base, possibly also the bases being themselves therapeutically active, for example with the same or similar activity as that of the esterifying component. In particular, it is possible to have hyaluronic esters deriving on the one hand from an antiinflammatory steroid, such as one of those mentioned previously, and on the other hand from a vitamin, from an alkaloid or from an antibiotic, such as one of those listed.
Method A:
The esters of hyaluronic acid may be prepared by methods known per se for the esterification of carboxylic acids, for example by treatment of free hyaluronic acid with the desired alcohols in the presence of catalyzing substances, such as strong inorganic acids or ionic exchangers of the acid type, or with an etherifying agent capable of introducing the desired alcoholic residue in the presence of inorganic or organic bases. As esterifying agents it is possible to use those known in literature, such as especially the esters of various inorganic acids or of organic sulphonic acids, such as hydracids, that is hydrocarbyl halogenides, such as methyl or ethyl iodide, or neutral sulphates or hydrocarbyl acids, alfites, carbonates, silicates, phosphites or hydrocarbyl sulfonates, such as methyl benzene or p-toluene-sulfonate or methyl or ethyl chlorosulfonate. The reaction may take place in a suitable solvent, for example an alcohol, preferably that corresponding to the alkyl group to be introduced in the carboxyl group. But the reaction may also take place in non-polar solventss, such as ketones, ethers, such as dioxane or aprotic solvents, such as dimethyl-sulphoxide. As a base it is possible to use for example a hydrate of an alkaline or alkaline earth metal or magnesium or silver oxide or a basic salt or one of these metals, such as a carbonate, and, of the organic bases, a tertiary azotized base, such as pyridine or collidine. In the place of the base it is also possible to use an ionic exchanger of the basic type.
Another esterification method employs the metal salts or salts with organic azotized bases, for example ammonium or ammonium substitute salts. Preferably, the salts of the alkaline or alkaline earth metals are used, but also any other metallic salt may be used. The esterifying agents are also in this case those mentioned above and the same applies to the solvents. It is preferable to use aprotic solvents, for example dimethylsulphoxide and dimethylformamide.
In the esters obtained according to this procedure or according to the other procedure described hereafter, free carboxylic groups of the partial esters may be salified, if desired, in a per se known manner.
Method B:
The hyaluronic esters may also be prepared by a method which consists of treating a quaternary ammonium salt of hyaluronic acid with an etherifying agent, preferably in an aprotic organic solvent.
As organic solvents it is preferable to use aprotic solvents, such as dialkylsulphoxides, dialklcarboxamides, such as in particular lower alkyl dialkylsulphoxides, especially dimethyl-sulphoxide, and lower alkyl dialkylamides of lower aliphatic acids, such as dimethyl or diethyl-formamide or dimethyl or diethylacetamide.
Other solvents however are to be considered which are not always aprotic, such as alcohols, ethers, ketones, esters, especially aliphatic or heterocyclic alcohols and ketones with a lower boiling point, such as hexafluoroisopropanol, trifluoroethanol, and N-methylpyrrolidone.
The reaction is effected preferably at a temperature range of between about 0xc2x0 C. and 100xc2x0 C., especially between about 25xc2x0 C. and 75xc2x0 C., for example at about 30xc2x0 C.
The esterification is carried out preferably by adding by degrees the esterifying agent to the above mentioned ammonium salt to one of the above mentioned solvents, for example to dimethyl-sulphoxide.
As an alkylating agent it is possible to use those mentioned above, especially the hydrocarbyl halogens, for example alkyl halogens. As starting quaternary ammonium salts it is preferable to use the lower ammonium tetraalkylates, with alkyl groups preferably between 1 and 6 carbon atoms. Mostly, hyaluronate of tetrabutylammonium is used. It is possible to prepare these quaternary ammonium salts by reacting a metallic salt of hyaluronic acid, preferably one of those mentioned above, especially sodium or potassium salt, in aqueous solution with a salified sulphonic resin with a quaternary ammonium base.
One variation of the previously described procedure consists in reacting a potassium or sodium salt of hyaluronic acid, suspended in a suitable solution such as dimethylsulphoxide, with a suitable alkylating agent in the presence of catalytic quantities of a quaternary ammonium salt, such as iodide of tetrabutylammonium.
For the preparation of the hyaluronic acid eaters, it is possible to use hyaluronic acids of any origin, such as for example the acids extracted from the above mentioned natural starting materials,, for example from cocks"" combs. The preparation of such acids is described in literature: preferably, purified hyaluronic acids are used. Especially used are hyaluronic acids comprising molecular fractions of the integral acids obtained directly by extraction of the organic materials with molecular weights varying within a wide range, for example from about 90%-80% (MW=11.7xe2x88x9210.4 million) to 0.2% (MW=30,000) of the molecular weight of the integral acid having a molecular weight of 13 million, preferably between 5% and 0.2%. Such fractions may be obtained with various procedures described in literature, such as by hydrolyzing, oxydizing, enzymatic or physical procedures, such as mechanical or radiational procedures. Primordial extracts are therefore often formed during these same by publication procedures (for example see the article by Balazs et al. quoted above in xe2x80x9cCosmetics and Toiletriesxe2x80x9d). The separation and purification of the molecular fractions obtained are brought about by known techniques, for example by molecular filtration.
Additionally useful are purified fractions obtainable from hyaluronic acid, such as for example the ones described in European Patent Publn. No. 0138572.
The salification of HY with the above metals, for the preparation of starting salts for the particular esterification procedure described above, is performed in a per se known manner, for example by reacting HY with the calculated base quantity, for example with alkaline hydrates or with basic salts of such metals, such as carbonates or bicarbonates.
In the partial esters it is possible to salify all the remaining carboxylic groups or only part of them, dosing the base quantities so as to obtain the desired stoichiometric degree of salification. With the correct degree of salification it is possible to obtain esters with a wide range of different dissociation constants and which therefore give the desired pH, in solution or xe2x80x9cin situxe2x80x9d at the time of therapeutic application.